This proposal is for an Exploratory/Developmental Grant Application (R21), specifically addressing RFA #PAR-01-047, the Cutting Edge Research Award (CEBRA) program. When opioid and adrenergic receptors are activated, pain transmission is inhibited, certainly in spinal cord dorsal horn by inhibition of release of excitatory transmitters from central afferent terminals and perhaps in skin by inhibition of transmission from epidermal nerve fiber terminals there. The proposed studies will determine whether 1) mu- and delta-opioid receptors (MOR and DOR) and alpha-2 adrenergic receptors (alpha-2A or alpha-2C AR) are colocalized in peripheral terminals of these nociceptive afferent fibers as in their central terminals, 2) their activation similarly results in analgesic effects, and 3) their co-activation yields a supraadditive interaction, synergy. Therapeutic exploitation of synergistic interactions in the periphery presents the opportunity to produce analgesia with minimal adverse effects mediated mostly in CNS (e.g., sedation, respiratory depression, hypotension, cognitive impairment, tolerance, dependence). The proposed research program will identify and characterize mechanisms through which and conditions under which both exogenous and endogenous spinally acting analgesic agents produce synergistic analgesia through the testing of the following hypotheses. HYPOTHESIS 1: alpha-2 AR and MOR or DOR are colocalized in nociceptive peripheral afferent terminals in skin. HYPOTHESIS 2: alpha-2 AR and OR activation inhibits afferent nociceptive activity in peripheral axons or terminals. HYPOTHESIS 3: alpha-2 AR and OR agonists interact synergistically when co-activated in peripheral terminals. These studies will be conducted in normal mice and mice subjected to inflammatory challenge. Electrophysiological studies will complement behavioral studies of peripheral analgesic action and synergy by determining the reduction of afferent activity induced by activation and co-activation of colocalized receptors. Testing the hypotheses will involve conduct of the following studies: 1) immunohistochemical determination of the presence and anatomical relationships between and among receptor subtypes in peripheral afferent terminals in naive vs. inflamed hindpaws; 2) behavioral and electrophysiological determination of analgesic effects and synergism upon co-administration of agonists selective for receptors MOR, DOR and alpha-2 AR; and 3) evaluation of the effect of antagonism of OR or deletion of alpha-2A or alpha-2C AR on the synergistic relationship between their respective agonists. The work is novel because alpha-2 AR localization in epidermal nerve fibers has not been characterized previously, AR-OR colocalization has not yet been studied and the functional consequences of activation or co-activation are unknown. The work is significant because bivalent targeting of analgesia to periphery may bypass centrally mediated side effects like sedation, tolerance, dependence and addiction liability.